Southwestern North America has large patches of relatively undisturbed desert that is increasingly impacted by roads, which can affect wildlife populations via roadkill and road avoidance. These road impacts may influence genetic variation by (1) limiting gene flow across roads and (2) decreasing genetic diversity in individuals nearest to roads. We tested the hypotheses that two-lane highways cause genetic differentiation and a decrease in genetic diversity in the desert kangaroo rat, Dipodomys deserti, and Merriam's kangaroo rat, D. merriami, at study sites in the Sonoran Desert of Arizona. We collected microsatellite data for both species from individuals distributed across the study sites. We found that (1) genetic differentiation across roads was minimal and generally insignificant for both species at both sites; and (2) genetic diversity was not significantly different near roads than far from them. These results were supported by Mantel tests that indicated little support for isolation-by-distance or isolation-by-barrier patterns. Our findings do not support the hypothesis that these highways have caused important changes in genetic patterns in these species over the time scale that the roads have been present. Our results suggest that partially permeable roads have negligible impacts on the genetic diversity and differentiation of kangaroo rat populations over the course of 40-50 years, but it also leaves open the possibility of such effects by impermeable barriers and/or larger time scales.